Project Summary The FOXO family of transcription factors are evolutionarily conserved regulators of homeostasis whose activities are linked to both increased lifespan and tumor suppression. Consistent with their role in maintaining cellular homeostasis, FOXO activity is upregulated by diverse types of cellular stress including nutrient/growth factor deprivation, DNA damage and oxidative stress. Control of FOXO activity is predominantly achieved through post- translational modifications that control nuclear-cytoplasmic shuttling of FOXO proteins. In the nucleus, FOXOs upregulate genes in multiple, often conflicting pathways including cell-cycle arrest, apoptosis, autophagy and ROS scavenger genes. How cells control FOXO activity to ensure that their response is appropriate for a given stress is an open question. To address this question we used CRISPR/Cas9 gene editing to fluorescently tag two FOXO proteins, Foxo1 and Foxo3a, at the endogenous locus of different cell lines. We use these lines to test the hypothesis that input/output specificity of the FOXO pathway is achieved through a dynamic control mechanism where different FOXO nuclear/cytoplasmic shuttling dynamics dictate separate cellular responses. Our hypothesis is inspired by similarities between the FOXO pathway and other transcription factors that use dynamic control mechanisms for input/output specificity including p53 and NF-?B. In addition, our preliminary data supports a role for FOXO dynamics in controlling cell fate. We found the single-cell dynamics of Foxo1 and Foxo3a shuttling change with different stimuli. Moreover, for the same stimulus we observed different dynamics for each isoform. In Aim 1 we explore the shuttling dynamics of Foxo1 and Foxo3a in response to serum starvation. We combine reverse phase protein arrays and RNA-seq to determine how time-dependent changes in key regulators control the dynamics of each isoform and how this is translated into different gene expression patterns. In Aim 2 we measure the dynamics of Foxo1 and Foxo3a shuttling as well as cell death in response to EGFR and Akt inhibitors. Previous experiments have shown that both Foxo1 and Foxo3a are required for cell death in response to EGFR inhibitors. We determine the dynamics of each isoform associated with cell death and develop transcriptional reporters to determine how dynamics are decoded by cells in terms of transcriptional output. In Aim 3 we develop an optogenetic system to control Foxo1 shuttling dynamics with light. We use this system to determine whether specific dynamic patterns of Foxo1 shuttling are sufficient to induce cell death and use RNA-seq to determine how changes in dynamics alter target gene expression. The experiments performed in this study will address a critical gap in our knowledge of how FOXO dynamics are controlled over time to enact specific outcomes. More broadly, our work will help elucidate how cell signaling circuits sense and respond to different signals.